The invention relates generally to tissue transplantation. More specifically, the invention relates to methods of increasing the replicative capacity of normally quiescent cells, such as normal somatic cells, by transient immortalization or transient telomerization, to produce cells suitable for cell therapy.
Cell therapy is an emerging field for the treatment of medical disorders. Cells from various tissue sources have been contemplated for transplantation into mammals, including human, recipients for treatment of disease or tissue or organ replacement. Use of primary cells for transplantation requires continuous access to fresh sources of tissue. This is problematic, however, particularly if human cells are desired. Normal human somatic cells display a finite replicative capacity of 50-100 population doublings characterized by a cessation of proliferation in spite of the presence of adequate growth factors. The replicative capacity can be considerably less (10-50 population doublings) when these cells are placed in culture in vitro. This cessation of replication in vitro is variously referred to as cellular senescence or cellular aging.
To generate tissue in sufficient quantity for therapeutic use, cells are commonly immortalized, to acquire unlimited replicative capacity and avoid cellular senescence. The identification of immortalizing genes and development of gene transfer methodologies permit generation of cell lines from cell types that are difficult to obtain in sufficient quantity or that have short lifetimes in culture. These immortalizing genes are typically generated by transfer of a virus or plasmid that contain an immortalizing gene. Cell immortalization increases the lifespan of a cell (especially in culture under replicative growth conditions), so that the resulting cell line is capable of being passaged many more times than the original primary cells.
The use of immortalized cells in cell therapy, however, can pose serious risks for patients, because immortalized cells are in many instances tumorigenic. Moreover, the exogenous DNA containing the nucleic acid capable of transforming the cells is commonly inserted into cells using infectious vectors, such as retroviral vectors. Virally infected cells also pose serious risks for patients, such as the potential of generating replication-competent virus during vector production; the potential recombination between the therapeutic virus and endogenous retroviral genomes, potentially generating infectious agents with novel cell specificities, host ranges, or increased virulence and cytotoxicity; and the potential independent integration into large number of cells, increasing the risk of tumorigenic insertional events.
Approaches to avoid these risks have focused on the removal of the genetic element when differentiation of the target cells is desired. One such approach involves the use of the Cre/loxP recombination system of bacteriophage P1. In the Cre/loxP procedure, the immortalizing gene, or oncogene, is flanked with recombinase recognition (loxP) sites for insertion, and subsequently removed via Cre-mediated deletion of the flanked gene segment. However, it is very difficult to prove the absence of residual immortalizing gene. Any leftover immortalizing gene would pose a serious risk to the recipient of the cell therapy, as it may allow these cells to continue to proliferate in the host after transplantation, and form a tumor. Accordingly, tissue from cells generated in this fashion is less desirable for cell therapy.
Thus, a need remains in the art for a method of cell proliferation that avoids the risks associated with the incorporation of exogenous immortalization genes in cells to be used for cell therapy.
The invention provides methods and compositions for generating cells that are not abundant, that are difficult to obtain in pure form in primary culture, that are in short supply (e.g., human cells), or that have brief lifetimes in culture. The invention relates to methods of conditional, transient cell proliferation in which immortalization or telomerization are initiated by the action of exogenously supplied molecules and terminated by the removal of these exogenously supplied molecules. Cells are proliferated in vitro for cell banking and, upon removal of the exogenous immortalizing or telomerizing fusion proteins, return to their non-proliferative state. The cells produced by the methods of the invention are suitable for transplantation and cell therapy. The methods of the invention can be used to proliferate any normally quiescent cell that can be induced to proliferate, such as normal somatic cells.
Provided in the invention are fusion proteins having a transport polypeptide amino acid sequence from herpesviral VP22, human immunodeficieny virus (HIV) TAT, or from homologues or fragments thereof, coupled to amino acid sequences from cell immortalization proteins. Examples of proteins or polypeptides for cell immortalization include the 12S and 13S products of the adenovirus E1A genes, SV40 small and large T antigens (subfragments and truncated versions), papilloma viruses E6 and E7, the Epstein-Barr Virus (EBV), Epstein-Barr nuclear antigen-2 (EBNA2), human T-cell leukemia virus-1 (HTLV-1), HTLV-1 tax, Herpesvirus Saimiri (HVS), mutant p53, and the proteins from oncogenes such as myc, c-jun, c-ras, c-Ha-ras, h-ras, v-src, c-fgr, myb, c-myc, n-myc, and Mdm2. Also provided in the invention are fusion proteins having a transport polypeptide amino acid sequence from herpesviral VP22, HIV TAT, or from homologues or fragments thereof, together with amino acid sequences from proteins or fragments thereof with telomerase-specific activity. Examples of proteins or fragments thereof with telomerase-specific activity include telomerase, p140, p105, p48, and p43.
In one embodiment of the invention, normally quiescent cells are transiently immortalized in order to proliferate these cells. Cells of interest are cultured in the presence of a fusion protein comprising a first polypeptide having the transport function of herpesviral VP22 protein or HIV TAT protein, and a second polypeptide having cell immortalization activity. The fusion protein is transported to the nucleus of the cells, and immortalizes the cells. The immortalized cells are expanded in the presence of the fusion protein. Once sufficient cells have been obtained, the fusion protein is removed from the growth medium, and the cells are cultured until they return to their original differentiated, non-immortalized state.
In another embodiment of the invention, normally quiescent cells are proliferated by transiently telomerizing these cells. Cells of interest are cultured in the presence of a fusion protein comprising a first polypeptide having the transport function of herpesviral VP22 protein, HIV TAT protein, and a second polypeptide having telomerase-specific activity. The fusion protein is transported to the nucleus of the cells where it synthesizes telomeric DNA at chromosomal ends, thereby preventing replicative senescence. The telomerized cells are expanded in the presence of the fusion protein. Once sufficient cells have been obtained, the fusion protein is removed from the growth medium, and the cells are cultured in a standard fashion in the absence of the exogenous immortalizing or telomerizing fusion proteins.
In yet another embodiment of the invention, the transporting protein (such as VP22 or TAT) and the immortalizing gene (such as hTERT, SV40, etc.) are mixed together and applied to the cells.
In still another embodiment of the invention, normally quiescent cells are proliferated by transiently telomerizing these cells. Cells of interest are cultured in the presence of two fusion proteins: (1) a fusion protein comprising a first polypeptide having the transport function of herpesviral VP22 protein or HIV TAT protein, and a second polypeptide having cell immortalization activity; (2) a fusion protein comprising a first polypeptide having the transport function of herpesviral VP22 protein or HIV TAT protein, and a second polypeptide having telomerase-specific activity. The cells may be cultured in the presence of more than one type of VP22-immortalization or TAT-immortalization fusion. The cells are expanded in the presence of the fusion proteins until sufficient cells with increased lifespan have been obtained. The fusion proteins are subsequently removed from the growth medium, and the cells are cultured until they return to their original differentiated, non-immortalized state.
The cells produced by the methods of the invention are not permanently immortalized (and thus are not tumorigenic or transformed) and are not virally infected. Accordingly, these cells, upon removal of the exogenous immortalizing or telomerizing fusion proteins, are suitable for transplantation and use in cell therapy.
In one embodiment of the invention, cellular immortalization is accomplished by direct transcriptional activation of telomerase reverse transcriptase, mediated by the addition of VP22-myc fusion protein. Wang, et al., 12 Genes and Dev. 1769-1774 (1998).
In another embodiment of the invention, a specific gene in a cell is transiently activated to specifically activate the expression of the endogenous gene of interest, and express the corresponding protein of interest. The proteins of interest include, but are not limited to, human growth hormone (hGH), erythropoietin (EPO), insulinotropin, insulin, leptin, hGCSF, Factor VIII, Factor VII, Factor IX, Factor X, and tissue-type plasminogen activator (tPA).